Serological investigation for West Nile virus, Anaplasma ovis and Leishmania infantum in Greek cattle Nektarios D. Giadinis1*, Panagiotis D. Katsoulos2, Dimosthenis Chochlakis2,3, Yannis Tselentis2,3, Pantelis Ntais2, Shawkat Q. Lafi4, Harilaos Karatzias1 & Anna Psaroulaki2

1 Clinic of Farm Animals, Faculty of Veterinary Medicine, University of , St. Voutyra 11, 546 27 Thessaloniki, . 2 Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University of Crete, Voutes‑Staurakia, 71110 Heraklion, Greece. 3 Regional Laboratory of Public Health of Crete, Voutes‑Staurakia, 71110 Heraklion, Greece. 4 Department of Pathology and Public Health, Faculty of Veterinary Medicine, Jordan University of Science and Technology, P.O. Box 3030, Irbid, Jordan. * Corresponding author at: Clinic of Farm Animals, Faculty of Veterinary Medicine, School of Health Sciences, Aristotle University, St. Voutyra 11, 54627 Thessaloniki, Greece. Tel.: +30 2310 994509, e‑mail:[email protected].

Veterinaria Italiana 2015, 51 (3), 205-209. doi: 10.12834/VetIt.174.523.4 Accepted: 19.04.2014 | Available on line: 30.09.2015

Keywords Summary Anaplasma ovis, This study investigates the seroprevalence of Greek adult cattle against West Nile virus, Cattle, Anaplasma ovis and Lehismania infantum. In total, 156 serum samples were examined, drawn Greek, from cattle between 2‑4 years old. All the examined cattle originated from slaughterhouses Lehismania infantum, of 4 prefectures in (Thessaloniki, , Chalkidiki, ), in 2 of which Seroprevalence, (Thessaloniki, Pella) human cases of West Nile virus had been recorded some months before. West Nile virus, Thirty out of the 156 (18.6%) samples have tested positive for West Nile virus and fifty‑five Zoonoses. (35.9%) samples for Anaplasma ovis. All the examined samples tested negative for Leishmania infantum. The prefectures with positive samples against West Nile virus also showed human cases of West Nile virus infections. This should raise questions whether cattle could become markers for West Nile virus activity in high risk areas.

Sieroprevalenza delle infezioni da West Nile virus, Anaplasma ovis e Leishmania infantum in bovini adulti nel Nord della Grecia

Parole chiave Riassunto Anaplasma ovis, Lo studio prende in esame le sieroprevalenze del virus della West Nile, di Anaplasma ovis e di Bovino, Lehismania infantum in bovini da 2 a 4 anni di età nel Nord della Grecia. Tra luglio e settembre Grecia, 2010 sono stati prelevati 156 campioni provenienti da macelli di 4 prefetture. In 2 di queste, Lehismania infantum, Salonicco e Pella, erano stati riportati, precedentemente a questa indagine, casi di West Nile Sieroprevalenza, nell’uomo. Trenta (18,6%) dei campioni analizzati sono risultati positivi al virus della West Nile e Virus della West Nile, 55 (35,9%) ad Anaplasma ovis. Tutti i 156 campioni sono risultati negativi a Leishmania infantum. Zoonosi. Le prefetture in cui sono stati prelevati i campioni positivi al virus West Nile sono risultate le stesse in cui il virus è stato isolato nell’uomo. Questo riscontro impone di considerare i bovini come indicatori dell’attività del virus in zone ad alto rischio.

Introduction infantum (Alam et al. 2011) can be found in cattle, which consequently can be regarded as potential reservoirs Cattle are regarded as a natural reservoir for some for leishmaniosis (at least in other parts of the world infectious diseases affecting livestock as well as such as ) (Singh et al. 2013) and anaplasmosis (de la humans (Muma et al. 2010). Infectious agents such Fuente et al. 2008). In the case of WNV, cattle may serve as Anaplasma spp. (Aktas et al. 2011) and Leishmania as sentinel of the presence of the pathogen in an area.

205 WNV, Anaplasma ovis, Leishmania infantum in greek cattle Giadinis et al.

To date, the epidemiological status of these IgG antibodies against WNV were tested using infections in cattle in Greece is unknown, although an ELISA commercial kit (ID screen West Nile these agents or their antibodies have been competition ELISA kit, ID.VET, Montpellier, France), recorded in other animal species or humans. according to the manufacturer’s instructions. West Nile infection has been recorded in humans, ELISA‑positive samples were confirmed by using chickens and wild birds (Chaskopoulou et al. 2011, the plaque reduction neutralization test (PRNT) Papa et al. 2011a, Papa et al. 2011b, Valiakos et al. (OIE 2013). 2011), and during 2010, human outbreaks with Due to possible cross‑reactions with other fatalities occurred (Chaskopoulou et al. 2011, flaviviruses, positive sera were also tested for tick Papa et al. 2011a). Anaplasma ovis has been found borne encephalitis virus (TBEV) [IMMUNOZIM® FSME to cause disease in small ruminants in Greece (TBE) IgG All Species, Progen Biotechnik, GmbH, (Giadinis et al. 2011), and possible cases in humans Heidelberg, ]. have also been reported (Chochlakis et al. 2010), at the same time Anaplasma phagocytophilum has The presence of IgG antibodies against A. ovis was been detected in Ixodes ricinus in Northern Greece performed using an ELISA commercial kit (VMRD, (Kachrimanidou et al. 2011). Disease caused by Washington, USA), modified to detectA. ovis as Leishmania infantum has already been diagnosed suggested by others (Scoles et al. 2008). in dogs (Koutinas et al. 2010) and humans (Diza Cattle sera were tested serologically using et al. 2008), while the spread of Phlebotomus spp. anti bovine, anti‑IgG antibodies, by Indirect all over Greece has been studied in the past (Ntais Immunofluorescence Test (IFAT, Leishmania SPOT et al. 2013). The objective of this preliminary study IF, Santa Cruz, Brazil). A series of two‑fold serum was to determine the prevalence of antibodies dilutions starting from 1/40 were performed. Based against WNV, A. ovis and L. infantum in dairy cattle on our experience the surveyed area is considered from Northern Greece. endemic for canine leishmaniosis, so a cut‑off titre of ≥ 1/160 was regarded as positive. Data were collected and entered in a data sheet Materials and methods and analyzed using IBM SPSS 20.0 software for The study was conducted from July to September Windows (IBM SPSS Corp., Armonk, NY, USA). 2010. Samples were taken from slaughterhouses Chi‑square (χ2) test, odd ration (OR) and the 95% of 4 prefectures in Northern Greece (Thessaloniki, confidence interval (CI) of the ORs were calculated Pella, Chalkidiki, Kilkis). In 2 of these prefectures to compare prevalence of antibodies against WNV, (Thessaloniki, Pella), 101 cases of WNV encephalitis A. ovis and L. infantum among the four study areas. had been diagnosed in humans during the The p‑value < 0.05 was considered statistically same summer (2010), while fewer similar cases significant. (Kachrimanidou et al. 2011) had also been diagnosed in the other 2 prefectures (Kilkis, Chalkidiki) (Muma et al. 2011). During the outbreak in the summer of Results 2010 in Central in Northern Greece, a Thirty out of 156 (18.6%) samples tested positive total of 197 patients with neuroinvasive disease for WNV. All sera positive for WNV were negative for was reported, of whom 33 (17%) died according to TBEV. Pella prefecture had significantly (p < 0.01) data of the Hellenic Centre for Disease Control and more positive samples compared to Thessaloniki 1 Prevention (Danis et al. 2011). (OR = 9.6; 95% CI = 3.02‑31.67), Chalkidiki In total, 156 blood samples were drawn before (OR = 15.71, 95% CI = 1.70‑117) and Kilkis (OR = 28.6, slaughter from adult cattle aged between 2 and 95% CI = 7.27‑136.62), whereas the prevalence 4 years. Fifty‑six samples were drawn from cattle was not significantly different among the others in Kilkis Prefecture, 54 samples in Thessaloniki prefectures (Table I). Table I shows also the WNV Prefecture, 34 in Pella Prefecture and 12 in Chalkidiki human cases recorded during 2010‑2011, while the Prefecture. Samples were kept refrigerated and the human cases and the cattle seropositivity to WNV in serum was separated by centrifugation at 2,500 x g the same span of time are depicted in Figure 1. for 20 min, approximately 1 hour after sampling. Fifty‑five out of 156 (35.9 %) samples were Sera were transferred in microcentrifuge tubes and tested positive for Anaplasma ovis. Chalkidiki stored at ‑20°C until analysis. prefecture had the highest prevalence, here the All samples were tested for antibodies against WNV, infection rate for A. ovis was significantly higher L. infantum and A. ovis. (p < 0.01) compared to the other 3 prefectures [(Thessaloniki OR = 2.6, 95% CI = 1.09‑6.23); (Kilkis OR = 11, 95% CI = 1.30‑82.91); (Pella OR = 33, 1 http://www.keelpno.gr/en‑us/home.aspx. 95% CI = 4.29‑156.71)]. Also, Thessaloniki had

206 Veterinaria Italiana 2015, 51 (3), 205-209. doi: 10.12834/VetIt.174.523.4 Giadinis et al. WNV, Anaplasma ovis, Leishmania infantum in greek cattle

Table I. Seroprevalence of West Nile virus (WNV), Anaplasma ovis (A. ovis) and Leishmania infantum (L. infantum) in greek cattle according to prefectures. Data on WNV human cases recorded during 2011-2012 originated from Hellenic Center for Disease Control & Prevention.

Animal population Human cases Prefecture Samples tested WNV positive (%) A. ovis positive (%) L. infantum positive WNV Thessaloniki 54 7 (13) 15 (27.8) 0 63 Pella 34 20 (58.8) 1 (2.9) 0 46 Chalkidiki 12 1 (8.33) 11 (91.67) 0 5 Kilkis 56 2 (3.57) 28 (50) 0 12 Total 156 30 (18.6) 55 (35.9) 0 126

Positive cattle Human WNV cases/ serum samples (%) 100.000 population WNV A. ovis 0-9 1-25 10-19 26-50 20-29 51-75 30-39 76-100 40-49

Figure 1. Human West Nile virus (WNV) cases and cattle WNV and Anaplasma ovis (A.ovis) seroprevalence in Northern Greece in 2010‑2011.

significantly higher prevalence (p < 0.05) compared of the disease were also detected. Therefore, cattle to Pella (OR = 12.69, 95% CI = 1.59‑82.67). could be regarded as natural markers (positive or Kilkis had significantly higher prevalence rate negative controls‑sentinel animals) for this virus in (p < 0.05) compared to Thessaloniki (OR = 2.6, high risk areas, as it already occurs for Bleutongue 95% CI = 1.09‑6.23) and to Pella (OR = 12.69, virus (Hoffmannet al. 2008). 95% CI = 1.59‑82.67) p< 0.01 (Table I). The high prevalence of the virus in these areas could Samples tested for L. infantum yielded negative results. be the result of intense agricultural production and especially of rice, a product that needs a lot of water, an excellent background for increased numbers of Discussion wild birds and mosquitoes that transmit WNV (Papa et al. 2011a). Lethal WNV encephalitis cases were reported in several mammalian species, including ruminants. Anaplasma ovis was detected in 35.9% of the In Northern Greece, human cases of the disease tested samples. Although in cattle the most have been reported in the past (Kecskeméti et al. pathogenic Anaplasma species are A. marginale and 2007, Papa et al. 2011a). Although the number of A. phagocytophilum (Durrani and Goyal 2012), A. ovis the samples tested was low, it is interesting that is the most commonly found Anaplasma in other the seroprevalence for WNV was 18.6%, a rate much ruminant species in Greece, it is potentially zoonotic higher than the one reported in a similar study in and could cause disease in cattle (Chochlakis et al. Turkey 6 years ago, in which a 4% seroprevalence 2010, Giadinis et al. 2011, Hornok et al. 2007, Hornok was detected (Ozkul et al. 2006). Most of the positive et al. 2012). samples (27 out of 30, 90%) of this study were In the present study, all the examined samples were recorded in the prefectures of Thessaloniki and found negative for L. infantum. Low leishmaniosis Pella, the 2 prefectures in which, most human cases seropositivity in cattle has been found in other

Veterinaria Italiana 2015, 51 (3), 205-209. doi: 10.12834/VetIt.174.523.4 207 WNV, Anaplasma ovis, Leishmania infantum in greek cattle Giadinis et al.

studies in Nepal (Bhattarai et al. 2010) and role in the maintenance and circulation of these 2 Bangladesh (Alam et al. 2011), countries that have pathogens in Northern Greece (Kachrimanidou et al. high prevalence of human leishmaniosis. 2011, Ntais et al. 2013), should act as a hint for the In conclusion, cattle from Northern Greece seem application of public health surveillance schemes. As to be negative to L. infantum, but they have a regards WNV, it could be suggested that cattle may remarkable positivity for WNV and A. ovis. With serve as sentinels in areas at risk for this pathogen. In regards to the latter, this means that they could any way, molecular studies would be very important serve as a possible hazard for the public health, as to confirm the results of the current survey and to well as for the livestock of the infected areas. Apart intensify the prophylactic measures (e.g. application from the results of the current study, the presence of proper and continuous disinfestation procedures) of vectors capable of transmitting and playing a in the herds.

References

Aktas M., Altay K. & Dumanli N. 2011. Molecular detection goats. In Proceedings of the 6th International Meeting and identification of Anaplasma and Ehrlichia species on Rickettsiae and Rickettsial Diseases, Crete, Greece. in cattle from Turkey. Ticks Tick borne Dis, 2, 62‑65. Hoffmann B., Sasserath M., Thalheim S., Bunzenthal C., Alam M.S., Ghosh D., Khan Md.G.M., Islam M.F., Mondal Strebelow G. & Beer M. 2008. Bluetongue virus serotype D., Itoh M., Islam Md.N. & Haque R. 2011. Survey of 8 reemergence in Germany, 2007 and 2008. Emerg domestic cattle for anti‑Leishmania DNA in a visceral Infect Dis, 14, 1421‑1423. leishmaniosis endemic area of Bagladesh. BMC Vet Hornok S., Elek V., De la Fuente J., Naranjo V., Farkas R., Res, 7, 27. Majoros G. & Földvári G. 2007. First serological and Bhattarai N.R., Van der Auwera G., Rijal S., Picado A., molecular evidence on the endemicity of Anaplasma Speybroeck N., Khanal B., de Doncker S., Lai Das ovis and A. marginale in Hungary. Vet Microbiol, 122, M., Ostyn B., Davies C., Coosemans M., Berkvens D., 316‑322. Boelaert M. & Dujardin,J.‑C. 2010. Domestic animals Hornok S., Micsutka A., Fernández de Mera I.G., Melil M.L., and epidemiology of visceral leishmaniosis, Nepal. Gönczi E., Tánczos B., Mangold A.J., Farkas R., Lutz H., Emerg Infect Dis, 16, 231‑237. Hofmann‑Lehmann R. & De la Fuente J. 2012. Fatal Chaskopoulou A., Dovas C.I., Chaintoutis S.C., Bouzalas bovine anaplasmosis in a herd with new genotypes of I., Ara G. & Papanastassopoulou M. 2011. Evidence Anaplasma marginale, Anaplasma ovis and concurrent of zoonotic circulation of West Nile virus (Nea haemoplasmosis. Res Vet Sci, 92 (1), 30‑35. Santa‑Greece‑2010, lineage 2), Greece, May to July Kachrimanidou M., Papa A., Chochlakis D., Pavlidou V. & 2011. Eurosurveill, 16, pii=19933. Psaroulaki A. 2011. Molecular evidence for Anaplasma Chochlakis D., Ioannou I., Tselentis Y. & Psaroulaki A. 2010. phagocytophilum in Ixodes ricinus ticks from Greece. Human anaplasmosis and Anaplasma ovis variant. Vector Borne Zoonotic Dis, 11, 1391‑1393. Emerg Infect Dis, 16, 1031‑1032. Kecskeméti S., Bajmócy E., Bacsadi A., Kiss I. & Bakonyi T. Danis K., Papa A., Theocharopoulos G., Dougas G., 2007. Encephalitis due to West Nile virus in a sheep. Vet Athanasiou M., Detsis M., Baka A., Lytras T., Mellou K., Rec, 161, 568‑569. Bonovas S. & Panagiotopoulos T. 2011. Outbreak of Koutinas A.F., Carlotti D.N., Koutinas C., Papadogiannakis West Nile virus infection in Greece, 2010. Emerg Infect E.I., Spanakos G.K. & Saridomichelakis M.N. 2010. Claw Dis, 17, 1868‑1872. histopathology and parasitic load in natural cases de la Fuente J., Ruiz‑Fons F., Naranjo V., Torina A., Rodríguez of canine leishmaniosis associated with Leishmania O. & Gortázar C. 2008. Evidence of Anaplasma infections infantum. Vet Dermatol, 21, 572‑577. in European roe deer (Capreolus capreolus) from Muma J.B., Munyeme M., Matope G., Siamudaala V.M., southern Spain. Res Vet Sci, 84, 382‑386. Munang’andu H.M., Matandiko W., Godfroid J., Skjerve Diza E., Kansouzidou A., Gerou S., Vezyri E., Metallidis E. & Tryland M. 2011. Brucella seroprevalence of the S. & Antoniadis A. 2008. Leishmaniases in Northern Kafue lechwe (Kobus leche kafuensis) and Black lechwe Greece: seroprevalence of the infection and incidence (Kobus leche smithemani): exposure associated to of the disease during the period 2001‑2006. Eur J Clin contact with cattle. Prev Vet Med, 100, 256‑260. Microbiol Infect Dis, 27, 997‑1003. Ntais P., Sifaki‑Pistola D., Christodoulou V., Messaritakis Durrani A.Z. & Goyal S.M. 2012. A retrospective study of I., Pratlong F., Poupalos G. & Antoniou M., 2013. Anaplasma in Minnesota cattle. Turk J Vet Anim Sci, Leishmaniases in Greece. Am J Trop Med Hyg, 89, 906‑915. 36, 131‑136. World Organisation for Animal Health (OIE). 2013. West Giadinis N., Chochlakis D., Tselentis Y., Petridou E., Nile fever, Chapter 2.1.20 Manual of diagnostic tests Karatzias H. & Psaroulaki, A. 2011. Acute and chronic and vaccines for terrestrial animals, OIE, Paris, France, 6. anaplasmosis due to Anaplasma ovis infection in dairy Ozkul A., Yildirim Y., Pinar D., Akcali A., Yilmaz V. & Colak,

208 Veterinaria Italiana 2015, 51 (3), 205-209. doi: 10.12834/VetIt.174.523.4 Giadinis et al. WNV, Anaplasma ovis, Leishmania infantum in greek cattle

D. 2006. Serological evidence of West Nile virus use in the diagnosis of A. ovis infections in domestic (WNV) in mammalian species in Turkey. Epidemiol sheep and Anaplasma spp. in wild ungulates. Vet Infect, 134, 826‑829. Microbiol, 130, 184‑190. Papa A., Xanthopoulou K., Gewehr S. & Mourelatos Singh N., Mishra J., Singh R. & Singh S. 2013. Animal S. 2011a. Detection of West Nile Virus lineage 2 in reservoirs of visceral leishmaniosis in India. J Parasitol, mosquitos during a human outbreak in Greece. Clin 99, 64‑67. Microbiol Infect, 17, 1176‑1180. Valiakos G., Touloudi A., Iacovakis C., Athanasiou L., Birtsas Papa A., Bakonyi T., Xanthopoulou K., Vásquez A., Tenorio A. & Nowotny N. 2011b. Genetic characterization of P., Spyrou V. & Billinis C. 2011. Molecular detection and west Nile Virus lineage 2, Greece, 2010. Emerg Infect Dis, phylogenetic analysis of west Nile virus lineage 2 in 17, 920‑922. sedentary wild birds (Eurasian magpie), Greece, 2010. EuroSurveill, 16, pii: 19862. Scoles G.A., Goff W.L., Lysyk T.J., Lewis G.S. & Knowles D.P. 2008. Validation of an Anaplasma marginale cELISA for

Veterinaria Italiana 2015, 51 (3), 205-209. doi: 10.12834/VetIt.174.523.4 209